Significance of the glutamic acid residues Glu334, Glu959, and Glu960 of the alpha subunits of Torpedo Na+, K+ pumps for transport activity and ouabain binding.
Glutamic acid residues in transmembrane segments of the alpha subunit of the Na+,K+-ATPase have been discussed as possible candidates for the binding sites of the transported cations. Here we report on effects of mutations of Glu334, Glu959, and Glu960 to alanine in ouabain-sensitive (OS) as well as ouabain-resistant (OR) ATPases of Torpedo electroplax expressed in Xenopus oocytes. All mutants are incorporated to about the same extend as the wild-type ATPases into the plasma membrane. None of the mutations produces complete inhibition of transport activity as judged from measurements of 86Rb+ uptake, membrane current, and ATPase activity. After conversion of OS to OR by mutation of the bordering residues of the first extracellular loop Gln118 to Arg and Asp129 to Asn, the Km value for inhibition by ouabain increases to 59 microM. Substitution of Glu334 to Ala in the OR pump variant restores ouabain sensitivity with a Km value of 0.12 microM, which is similar to that of the endogenous Xenopus pump. After substitution of Glu960 by Ala in the OR pump, ouabain sensitivity is partially restored. The Km values for pump stimulation by external K+ appear to be reduced in the OR compared to the OS pump. Mutation of Glu959 and Glu960 to Ala has no pronounced effects on the potential-dependent Km values at external pH 7.8; only in the Glu959-mutated OR pump, the apparent Km at 0 mV is raised. We conclude that none of the mutated glutamic acid residues is essential for cation coordination, but that GIu334, and in part also Glu960, seems to be involved in preserving the ouabain-resistant conformation of the enzyme.
Vasilets
L A
LA
Max-Planck Institut für Biophysik, Frankfurt/Main, Germany. 100304.1621@compuserve.com
Takeda
K
K
Kawamura
M
M
Schwarz
W
W
D000818:Animals; D018698:Glutamic Acid / Q000378:metabolism*; D016623:Ion Pumps; D017136:Ion Transport; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014101:Torpedo; D014982:Xenopus laevis
D016623:Ion Pumps; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium